Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile associated disease are substantial and increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of modifying multiple targets within eukaryotic host cells. The toxins contain four functional domains that are associated with host-receptor binding, delivery across a membrane, autoprocessing, and the enzymatic inactivation of host Rho-proteins. The interaction with receptors, the kinetics of delivery, the ease of autoprocessing, and the specificity for cellular substrates are affected by sequence variation among different toxin isoforms and distinct environmental conditions within the host cell. The central objective of the proposed research program is to elucidate structural and molecular mechanisms of toxin function. We propose a hybrid approach that combines cryo-electron microscopy (cryo-EM), X-ray crystallography, biochemical and cell-based functional studies. In Aim 1, we will elucidate a structure of the TcdA holotoxin using cryo-EM and probe the multiple conformational stages that are accessed as the toxin is exposed to host environmental cues (low pH, inostol-6-phosphate and reductant). In Aim 2, we will determine the X-ray crystal structure of the TcdA delivery domain and identify the sequences that are associated with membrane pore formation. In Aim 3, we will compare the glucosyltransferase properties of TcdA to those of TcdB. A mechanistic understanding of the conserved and divergent features of these two toxins will provide a necessary platform for therapeutic inhibitor design.